guest ::
| Home > Publications database > Development of a transcriptional biosensor and reengineering of its ligand specificity using fluorescence-activated cell sorting |
| Home > Publications database > Development of a transcriptional biosensor and reengineering of its ligand specificity using fluorescence-activated cell sorting |
| Book/Dissertation / PhD Thesis | FZJ-2019-05198 |
2020
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
ISBN: 978-3-95806-515-4
Please use a persistent id in citations: http://hdl.handle.net/2128/26583 urn:nbn:de:0001-2021080228
Abstract: Important chemical compounds of our daily life such as amino acids, antibiotics or vitamins are produced by microorganisms at large‐scale. Also, there is growing interest in the microbial synthesis of many other compounds including pharmaceutically interesting secondary metabolites from plants. However, development and improvement of the microbial producer strains is time‐consuming and cost‐intensive. In this context,biosensor‐based fluorescence‐active cell sorting (FACS) to identify suitable production strain variants representsa promising approach to tackle these challenges. In this dissertation, the application of transcription factor‐based biosensors in combination with FACS for high-throughput screening of enzyme libraries was investigated in $\textit{Escherichia coli}$. Furthermore, the construction of biosensors with modified ligand spectrum from an existing biosensor was pursued to expand the repertoire of biosensor‐detectable substances. Initially, the transcription factor‐based biosensor pSenCA which can be used to convert cytosolic concentrations of the phenylpropanoid trans‐cinnamic acid (CA) to a fluorescence output signal, was constructed and characterized. The biosensor is composed of the transcriptional regulator HcaR from $\textit{Escherichia coli}$ and its target promoter P$_{hcaE}$, transcriptionally fused with the $\textit{eyfp}$ gene encoding an autofluorescent protein. This biosensor was subsequently used to optimize an L‐phenylalanine/L‐tyrosine ammonia lyase from $\textit{Trichosporon cutaneum}$ (Xal$_{Tc}$), by a directed evolution approach. Aromatic amino acid ammonia lyases represent the key enzyme in many plant polyphenol biosynthetic pathways. The use of an expression system with titratable expression strength of the ammonia lyase gene as well as a significant reduction of the initial cell density priorto screening were prerequisites for an effective isolation of CA producers from mixed cultures with nonproducers. The established screening method was subsequently used to screen a randomly mutagenized ammonia lyase library of 2.4×10$^{6}$ variants for improved fluorescence. All 182 clones isolated by FACS were CA producers, 138 produced at least 10 % more CA compared to the parent strain. The best strain showed a 60 % increase in CA production. Seven Xal$_{Tc}$ variants investigated $\textit{in vitro}$ exhibited up to 12 % increased specific activity and up to 20 % increased substrate affinity. In the second project, 15 amino acids in the ligand binding site of the regulator protein HcaR, which were identified by $\textit{in silico}$ structure analysis, were randomized by site saturation mutagenesis. The resulting HcaR biosensor libraries were screened for variants with increased specificity for 3,5‐dihydroxyphenylpropionate using FACS. These experiments resulted in the isolation of pSenGeneral, a sensor variant with a significantly broadenedligand spectrum. In a second round of biosensor evolution, additional libraries based on pSenGeneral were constructed and screened for variants with specificity for various compounds of biotechnological interest. As a result, biosensor variants for the detection of 4‐hydroxybenzoic acid, 6‐methylsalicylate, $\textit{p}$‐coumaric acid, or 5‐bromoferulic acid could be isolated. In the future, these newly designed biosensors for small aromatic compounds could find an application during microbial strain development and might represent a good starting point for the development of additional biosensors for other aromatic molecules of biotechnological interest.
; 
|
The record appears in these collections: |
PDF
Fulltext by OpenAccess repository